Cellular mechanics and intracellular organization

Mechanical signals affect and regulate many aspects of the cell behaviour, including growth, differentiation, gene expression and cell death. This thesis investigates the manner by which mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, a combined fluorescence-atomic force microscope (AFM) was used to produce well defined nanomechanical perturbations while simultaneously tracking the real-time motion of fluorescently labelled intracellular organelles in live cells. By tracking instantaneous displacements of mitochondria far from the point of indentation, insights can be gained into the long-distance propagation of forces and the role of the cytoskeleton in force transmission. Quantitative analysis and tracking of mitochondria, using several image registration and tracking techniques, revealed an increase of approximately 40% in the mean mitochondrial displacement following AFM perturbation. Furthermore, when either the actin cytoskeleton or microtubules were disrupted using anti-cytoskeletal drugs, no significant change in mitochondrial displacement was observed following indentation, revealing the crucial role of both cytoskeletal networks in the long-distance transmission of forces through the cell. In addition, the effect of retinol and conjugated linoleic acid (CLA), compounds that have diverse effects on various cellular processes, on the mechanical behaviour of the cell was examined: both compounds were found to have a significant detrimental effect on the formation of focal adhesions, which was directly correlated to the measured cell elasticity (Young’s modulus) of the cell. Furthermore, quantification of mitochondrial displacements in response to applied AFM perturbations showed force propagation through the cytoskeleton to be blunted. Treatment of the two compounds in combination showed an additive effect. These results may broaden our understanding of the interplay between cell mechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease

Tracking displacements of intracellular organelles in response to nanomechanical forces

The living cell is under constant influence of mechanical forces from its environment. These forces affect many aspects of the cell's behaviour, including morphology, growth, cell differentiation, protein synthesis and cell death. In this study we show how mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, we used a combined fluorescence-atomic force microscope (AFM) to produce nanomechanical perturbations while simultaneously tracking the real-time motion of fluorescently labelled mitochondria in live cells. Feature point tracking was then used to analyze and quantify the structural displacements. Following indentation from the AFM tip, the average mitochondrial displacement showed an increase of similar to 40% in comparison to the natural movement. These results show how mitochondrial structures that are far away from the point of force (up to similar to 40 mu m) are instantaneously affected by extracellular perturbations

Development of a Combined Atomic Force Microscopy and Side-View Imaging System for Mechanotransduction Research

Key metrics for understanding cell response to mechanical stimuli include rearrangement of the cytoskeletal and nucleoskeletal structure, induced strains and biochemical distributions; however, structural information during applied stress is limited by our ability to image cells under load. In order to study the mechanics of single cells and subcellular components under load, I have developed a unique imaging system that combines an atomic force microscope (AFM) with both vertical light-sheet (VLS) illumination and a new imaging technique called PRISM – Pathway Rotated Imaging for Sideways Microscopy. The combined AFM and PRISM system facilitates the imaging of cell deformation in the direction of applied force with accompanying pico-Newton resolution force measurements. The additional inclusion of light-sheet microscopy improves the signal-to-noise ratio achieved by illumination of only a thin layer of the cell. This system is capable of pico-newton resolution force measurements with simultaneous side-view high frame rate imaging for single-molecule and single-cell force studies. Longer-term goals for this instrument are to investigate how external mechanical stimuli, specifically single-molecule interactions, alter gene expression, motility, and differentiation. The overall goal of my dissertation work is to design a tool useful for mechanobiology studies of single cells. This requires the design and implementation of PRISM and VLS systems that can be coupled to the standard Asylum AFM on inverted optical microscope. I also examine the strategy and implementation of experimental procedures and data analysis pipelines for single-cell and single-molecule force spectroscopy. These goals can be broken down as follows: • Performed single-molecule force spectroscopy experiments. • Performed single-cell force spectroscopy experiments. • Constructed and characterized the side-view microscopy system. • Applied combined AFM and side-vew microscopy system.Doctor of Philosoph